Protein function can be modified and improved in vitro by a variety of methods, including site-directed mutagenesis, combinatorial cloning and random mutagenesis combined with an appropriate selection system.
The method of random mutagenesis together with selection has been used in a number of cases to improve protein function and generally follows one of two strategies. The first involves randomisation of the entire gene sequence in combination with the selection of a variant (mutant) protein with desired characteristics. This process can be repeated on the selected variant until a protein variant is found which is considered optimal. Mutations are typically introduced by error-prone PCR (Leung et al., 1989, Technique, 1:11-15) with a mutation rate of approximately 0.7%. The second strategy is to mutagenize defined regions of the gene with degenerate primers (“saturation mutagenesis”), which allows for mutation rates of up to 100% (Griffiths et al., 1994, EMBO. J, 13:3245-3260; Yang et al., 1995, J. Mol. Biol. 254:392-403), followed by selection of variants with interesting characteristics. The mutated DNA regions from different variants, each with interesting characteristics, may subsequently be combined into one coding sequence (Yang et al., ibid).
Another process for in vitro mutation of protein function is “DNA shuffling,” which uses random fragmentation of DNA and assembly of fragments into a functional coding sequence (Stemmer, 1994, Nature 370:389-391). The DNA shuffling process generates diversity by recombination, combining useful mutations from individual genes. The genes are randomly fragmented using DNase I and then reassembled by recombination with each other. The starting material can be either a single gene (first randomly mutated using error-prone PCR) or naturally occurring homologous sequences (so-called family shuffling).
V(D)J recombination is the process responsible for the assembly of antibody gene segments (V, D and J; or V and J in the case of the light chain) and as part of the assembly process creates the CDR3 of the respective antibody chain. V(D)J recombination can be considered conceptually as a segment shuffler for antibodies, i.e. it brings together the different VH segments, D segments and JH segments to create an antibody (similarly V(D)J recombination at the light chain assembles different combinations of light chain V and J segments at either the kappa or lambda locus). The recombination event results in large chromosomal deletions in order to bring the required segments together. V(D)J recombination is targeted by the presence of specific DNA sequences called the recombination signal sequences (RSSs). The recombination reaction involves the recombination proteins RAG-1 and RAG-2 and follows a 12/23 rule where an RSS with a 23 bp spacer is paired only with an RSS with 12 bp spacer and adjacent sequences are subsequently joined by double-stranded break repair proteins.
The V(D)J recombination reaction is responsible for the creation of CDR3, as it is the sole mechanism for gene segment assembly and antibody generation in the bone marrow. V(D)J recombination does not occur at CDR1 or CDR2. V(D)J recombination therefore is not involved in affinity maturation but in primary B cell development and antibody assembly.
U.S. Pat. No. 8,012,714 describes compositions and methods for generating sequence diversity in the CDR3 region of de novo generated immunoglobulins in vitro. The methods comprise constructing nucleic acid molecules that comprise polynucleotide sequences encoding immunoglobulin V, D, J and C regions, together with recombination signal sequences (RSS), and subsequently introducing these nucleic acid molecules into suitable recombination-competent host cells. The methods provide for the assembly of gene segments to generate a functional antibody in vitro.
The use of “protein scaffolds” for the generation of novel binding proteins via combinatorial engineering has recently emerged as a powerful alternative to natural or recombinant antibodies. It has been found that novel binding sites can be introduced into proteins from several protein families with non-Ig architectures by combinatorial engineering, such as site-directed random mutagenesis combined with phage display or other selection techniques (Rothe, A., et al., 2006, FASEB J., 20:1599-1610). This concept requires a stable protein architecture (“scaffold”) tolerating multiple substitutions or insertions at the primary structural level (see reviews by Binz, H. K., et al., 2005, Nature Biotechnology, 23(10):1257-1268; Nygren, P-A. & Skerra, A., 2004, J Immunol. Methods, 290:3-28, and Gebauer, M. & Skerra, A., 2009, Curr. Op. Chem. Biol., 13:245-255).
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.